Tissue procurement by rapid autopsies provides an effective way to investigate tumor biology of primary and a broad range of metastatic sites in a manner not possible by any other means. In addition to tumor heterogeneity, tissue obtained at autopsy could yield important biologic insights into frequency and nature of secondary mutations associated with tumor progression, novel biomarkers and mechanisms of resistance to treatment. The full extent and consequences of tumor heterogeneity can be evaluated by deep sequencing and global analysis of genetic alterations at the protein level of simultaneous core biopsies from several areas of the primary tumor and metastases and correlation with clinical outcome. To our knowledge, no such studies have been done in NSCLC. We intend to collect tumor tissue from up to ten different sites of metastatic disease to study inter-tumor heterogeneity. Up to six different cores from each site will be stored properly to interrogate intra-tumor heterogeneity. Next generation sequencing and in-depth mass spectrometry-based proteomics analyses will be performed on these samples to assay tumor heterogeneity. We will also attempt to generate cell lines from several sites from a patient that are expected to be unique reagents to study tumor heterogeneity and tumor biology. We have so far performed rapid autopsies on four lung adenocarcinoma, one mesothelioma patient, and one thymic carcinoma patient. We have collected tumor and normal tissues from all possible sites of disease. Multiple cores have been collected from each site of disease. We have also been successful in generating cell lines from tumor tissue of two of these patients. We have completed 52 exome and 30 transcriptome sequencing of a subset of these samples among these six patients. We have analyzed this data. We have uncovered significant heterogeneity in SNV and CNV specific to each patient. Most improtantly, we have discovered that APOBEC-mediated mutagenesis was a major driver of heterogeneity in specific patients. We have also performed mass spectrometry-based proteomics analysis on a seuset of these rapid autopsy samples. We are now correlating the genomic and proteomic heterogeneity (both intra-tumor and inter-tumor) in our cohort of rapid-autopsy patients.